Simultaneous blockade of the CD28 and CD40 co-stimulatory pathways promotes prolonged survival of skin allografts and xenografts. However, in the long-term the majority of these skin grafts fail over a period of several weeks. This late failure appears to occur via CD4/CD28 independent mechanisms, as prolonged costimulation blockade does not prevent late graft loss. The overall goal of this project is to define the mechanisms involved in costimulation blockade-resistant rejection of skin allografts in mice. We will focus primarily on the most difficult allogeneic combination, BALB/c to B6, in which the co-stimulatory blockade resistant-rejection is most prominent. Our central hypothesis is that CD8+ T cells and possibly NK cells play a pivotal role in co-stimulation blockade-resistant rejection. Furthermore, we hypothesize that 4-1BBL or fasL are important accessory molecules in this process. We believe that by understanding the relevant cells and signaling pathways in these mice we can develop additional strategies to more effectively inhibit allograft rejection. The specific aims of this project are: (1) To determine which lymphocyte subsets contribute to costimulation blockade-resistant rejection; 92) To determine if alternative co-stimulatory mechanisms contribute to CD40 and CD28 blockade-resistant rejection, with particular emphasis on the 4-1BB co- stimulatory pathway; and (3) To determine the role of fas ligand in costimulation blockade-resistant rejection. We will use the knowledge gained from these experiments to direct our efforts to optimize costimulation pathway blockade strategies to induce transplantation tolerance. These studies will first be performed first in mice and then in primates in Project #3 of this Program Project grant.